Gram-negative pathogens producing metallo-?-lactamases, MBLs, seriously threaten the public health. MBLs are the most worrisome carbapenemases, inactivating the ?last resort? carbapenems and most ?-lactams, and resist all commercially available ?-lactamase inhibitors (BLIs). The main challenges in MBL inhibitor design are understanding the reaction mechanism as it relates to the structural diversity of the 3 distinct subclasses (B1, B2, and B3). In the previous 5-year funding cycle, we achieved important milestones: i) identified the active form of clinically relevant MBLs in the bacterial periplasm; ii) characterized NDM-1 as a membrane-bound protein, establishing how this localization endows NDM-1 with unique stability; iii) demonstrated that MBLs of all 3 subclasses utilize a common mechanism for carbapenem hydrolysis suggesting novel approaches for inhibitor development; iv) designed a series of novel compounds, bisthiazolidines (BTZs), as substrate mimics, comprising a non- ?-lactam ?penicillin core? decorated with metal binding groups; and v) showed that BTZs are non-toxic, effective cross-class MBL inhibitors and identified the structural bases of their inhibitory action. Responding to the clear urgency to find novel therapies, our team will build on these accomplishments to identify, synthesize, evaluate and develop new cross-class MBL inhibitors. Our unique approach is based upon a mechanistic understanding of MBL catalysis which will be utilized to inspire potent inhibitors. To this end, we will synthesize new compounds as mimics of mechanistic intermediates or product mimics [Thiazolidines (TZs), ?4- Thiazolidines (?4-TZs), and ?4-Oxazolidines (?4-OXZs)] or carbapenem mimics [?4-Bisthiazolidines (?4-BTZs) and Bicyclooctanes (BCOs)]. Our second specific aim will evaluate inhibitors for in vitro activity against MBLs of all subclasses. We will next assay the impact of inhibitors in potentiating ?-lactam efficacy against MBL- producing model strains, assess differences between in vitro assays and effect on bacteria, and validate the selected inhibitors against a panel of clinical strains with different MBL alleles. Our third aim will combine NMR and X-ray crystallography to study the structure of MBL-inhibitor adducts aimed to provide details for inhibitor improvement. We will also pursue mechanistic studies using micro-focusing spectroscopy and crystallography coupled to XFEL (X-ray free electron lasers) to trap transient ?-lactam-bound species in the enzymes NDM-1, L1, and VIM. This ?high-risk, high impact? innovative approach using new technologies will provide information for inhibitor improvement. Lastly, we will assay off-target activity and in vitro toxicity of the synthesized compounds, perform time-kill assays for meropenem-BLI combinations in clinical strains; and use mouse blood stream and lung infection models to assess the in vivo potency of meropenem/MBL-inhibitor combinations. This knowledge will serve to inform the design of therapeutic leads to combat MBL producing bacteria.